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Team:DTU-Denmark-2/Team/Protocols
From 2011.igem.org
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'''Cell culture and reagents''' | '''Cell culture and reagents''' | ||
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HeLa and U2OS cells were kindly provided by The Danish Cancer Society. HeLa cells are a spontaneously immortilized cell line derived from cervical cancer cells taken from Henrietta Lacks, a patient that died of cancer in 1951. The HeLa cell line is the oldest and most commonly used human cell line, e.g. the cells were used to test the first polio vaccine in the 1950s, and since then they have been used for research in cancer, AIDS, etc. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology. | HeLa and U2OS cells were kindly provided by The Danish Cancer Society. HeLa cells are a spontaneously immortilized cell line derived from cervical cancer cells taken from Henrietta Lacks, a patient that died of cancer in 1951. The HeLa cell line is the oldest and most commonly used human cell line, e.g. the cells were used to test the first polio vaccine in the 1950s, and since then they have been used for research in cancer, AIDS, etc. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology. | ||
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'''Medium''' | '''Medium''' | ||
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500 ml DMEM | 500 ml DMEM | ||
50 ml Fetal Calf Serum (FCS) | 50 ml Fetal Calf Serum (FCS) | ||
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5 ml Streptomycin | 5 ml Streptomycin | ||
| - | + | Add FCS, penicillin, and streptomycin to a new flask of DMEM. Store at 5°C. | |
| - | + | ||
'''Cultivation of the cells''' | '''Cultivation of the cells''' | ||
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25 cm2 culture flask | 25 cm2 culture flask | ||
Complete DMEM | Complete DMEM | ||
| - | The HeLA and the U2OS cells are kept at -80°C until defrosted gently at 37° | + | Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before).The HeLA and the U2OS cells are kept at -80°C until defrosted gently at 37°. Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette. The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded. The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks. |
| - | Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette. The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded. The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks. | + | |
'''Passing and maintenance of HeLa and U2OS cells''' | '''Passing and maintenance of HeLa and U2OS cells''' | ||
Revision as of 08:08, 15 August 2011
Contents |
Protocols
Amplifying of biobricks by PCR
PCR MIX
| PCR mix | 1 x PCR mix á 50µl |
|---|---|
| 5 x HF PCR buffer with MgCl2 or GC buffer | 10µl |
| dNTP’s 2mM | 5µl |
| Primer forward 10 µM | 4µl |
| Primer reverse 10 µM | 4µl |
| Phusion DNA polymerase 5u/µl | 0.3µl |
| DNA template | 0.5µl |
| MilliQ water | 26.20µl |
PCR Programs
| Temperature [°C] | Time [min] | Cycles |
|---|---|---|
| 98 | 2:00 | |
| 98 | 0:10 | 35 |
| 59 | 0:30 | - |
| 72 | 3:00 | - |
| 72 | 5:00 | |
| 12 | Store |
USER cloning
Tranformation in E.coli
Purifying of plasmid
Fungi
Transformation in fungi
Genetic Transformation of filamentous fungi – protocol from Center for Microbial Biotechnology (CMB) at DTU, Author Nielsen, J. B.
Media
- Minimal medium (MM) (1L) -50 mL D-glucose 20% w/V, 20 mL 50x mineral mix, 10 mL 1 M sodium nitrate, 20 g ager.
- Transformation media(TM)(1L)- 342.3 g Sucrose, 20 mL 50x mineral mix, 20 g agar.
- Mineral Mix (1L)- 26g KCL, 26g MgSO4·7H2O, 76g KH2PO4, 50 mL Trace element solution, MIlli-Q water to volume 1000 mL
- D-glucose 20% (0.5 L) - 100g D-glucose and MilliQ water up to 500 ml
- Aspergillus protoplastationbuffer (APB)- Final conc. 1.1 M MgSO4 and 10 mM Na-phosphate buffer. pH is adjusted with 2 N NaOH to 5.8.
- Aspergillus transformation buffer (ATB)- Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.
- PCT (200ml) - Final conc: 50% w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store at 4 °C
Initiation: The host strain is grown as three-point stabs on Minimal medium plates with the require suppliants added. MM will for convenience throughout the protocol refer to MM with the supplements included.
Inoculation: The conidia are harvest by adding 5 ml of MM and firmly rub with a sterile Drigalsky spatula. The conidial suspension is pipette to a sterile 500 ml shake flask containing 100 ml MM. The cultures are incubated at 30 °C with 150 rpm of shaking over night (14-20 hours)
Mycelial harvest: A funnel with a sterile Mira cloth (filter) is used to harvest mycelia. To remove residual glucose from mycelia the biomass are wash with Aspergillus protoplastationbuffer (APB). The filtered biomass is transferred to a new Falcon tube with a sterile spoon.
Protoplastation: Mycellium is resuspenden in 10 ml filter-sterillized(0.45μm filters) APB containing 40 mg Glucanex/ml. The Glucanex is dissolved in APB with gentle magnetic stirring less than 100/min. Mycelia with dissolved Glucanex are mixed at 30 °C with 150 rpm of shaking for 2-3 hours.
Portoplast solutions are diluted in APB adding up to 40 ml mark. An overlay of max. 5ml Aspergillus transformation buffer (ATB) diluted to ½x with sterile MilliQ-water is carefully placed on top of the APB. Centrifuged at 13 min 3000 RCF in Sorvall centrifuge. Protoplates should be observed as a halo of with slurry in the interphase of the two liquids. Withdraw of the protoplasts are done with pipette and placed in a Falcon tube. ATB is added up to 40 ml mark. Centrifuge at 300 RCF in 13 min and supernatanted are discarded.
The protoplastes are resuspended in 1 ml ATB with a 5 ml pipette.
Genetic transformation: Aliquotes of 200 μl are transferred to a 1.5 ml Eppendorf tube containgen the DNA for transformation. DNA concentration with bipartite substrates should be 2x(1-5μl). Protoplast and DNA are incubated at room temperature for at least 30 min.
Protoplat and DNA suspension are added to 1 ml PCT in a 15 ml tube and shake gently. Incubated for 1-5 min at room temperature. Diluted in 3 ml ATB. The tube is filled with molten transformation medium (TM) agar (temperature of 40-45°C) to apporeimately 12 ml. The tube is mix rapidly by inverting the tube twice. Poured directly on pre-made TM plates and incubated at 37°C for 3-8 days.
Passing and maintenance of mammalian cells
Cell culture and reagents
HeLa and U2OS cells were kindly provided by The Danish Cancer Society. HeLa cells are a spontaneously immortilized cell line derived from cervical cancer cells taken from Henrietta Lacks, a patient that died of cancer in 1951. The HeLa cell line is the oldest and most commonly used human cell line, e.g. the cells were used to test the first polio vaccine in the 1950s, and since then they have been used for research in cancer, AIDS, etc. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology.
The cells are cultivated in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Penicillin, Streptomycin, and 10 % heat-inactivated foetal calf serum (FCS). Penicillin and Streptomycin are added to prevent any microbial growth. FCS is added to supply essential non-defined components, such as serum proteins and lipids. Supplemented DMEM medium is referred to as complete DMEM throughout the report. The cells are adherent and are kept in 75 cm2 culture flask until 80-100% confluency, where they are passed on to new culture flasks or cover slips.
Medium
500 ml DMEM 50 ml Fetal Calf Serum (FCS) 5 ml Penicillin 5 ml Streptomycin
Add FCS, penicillin, and streptomycin to a new flask of DMEM. Store at 5°C.
Cultivation of the cells
25 cm2 culture flask Complete DMEM
Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before).The HeLA and the U2OS cells are kept at -80°C until defrosted gently at 37°. Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette. The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded. The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until the following day, where they are passed on to larger culture flasks.
Passing and maintenance of HeLa and U2OS cells
Procedure for cells in 75 cm2 culture flask: 75 cm2 culture flask 50 ml vial 1, 2, 5, 10, 25 ml pipettes Complete DMEM medium 0.05 % EDTA-trypsin
The appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells. The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use. 1 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly. 1 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2. 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them. 2,5 ml (depends on the concentration of the cells) cell suspension is transferred to a new 75 cm2 culture flask. The cells are kept at 37°C in a 5% CO2 incubator until they are 80-100% confluent. This takes about 2-3 days. Then they are passed on to a new flask or plate.
